Tumor necrosis factor-inducible gene 6 promotes liver regeneration in mice with acute liver injury

Sihyung Wang, Ji-Seon Lee, Jeongeun Hyun, Jieun Kim, Seung U Kim, Hyuk-Jin Cha, Youngmi Jung

Tumor necrosis factor-inducible gene 6 protein (TSG-6), one of the cytokines released by human mesenchymal stem/stromal cells (hMSC), has an anti-inflammatory effect and alleviates several pathological conditions; however, the hepatoprotective potential of TSG-6 remains unclear. We investigated whether TSG-6 promoted liver regeneration in acute liver failure. The immortalized hMSC (B10) constitutively over-expressing TSG-6 or empty plasmid (NC: Negative Control) were established, and either TSG-6 or NC-conditioned medium (CM) was intraperitoneally injected into mice with acute liver damage caused by CCl4. Mice were sacrificed at 3 days post-CM treatment. Higher expression and the immunosuppressive activity of TSG-6 were observed in CM from TSG-6-hMSC. The obvious histomorphological liver injury and increased level of liver enzymes were shown in CCl4-treated mice with or without NC-CM, whereas those observations were markedly ameliorated in TSG-6-CM-treated mice with CCl4. Ki67-positive hepatocytic cells were accumulated in the liver of the CCl4 + TSG-6 group. RNA analysis showed the decrease in both of inflammation markers, tnfα, il-1β, cxcl1 and cxcl2, and fibrotic markers, tgf-β1, α-sma and collagen α1, in the CCl4 + TSG-6 group, compared to the CCl4 or the CCl4 + NC group. Protein analysis confirmed the lower expression of TGF-β1 and α-SMA in the CCl4 + TSG-6 than the CCl4 or the CCl4 + NC group. Immunostaining for α-SMA also revealed the accumulation of the activated hepatic stellate cells in the livers of mice in the CCl4 and CCl4 + NC groups, but not in the livers of mice from the CCl4 + TSG-6 group. The cultured LX2 cells, human hepatic stellate cell line, in TSG-6-CM showed the reduced expression of fibrotic markers, tgf-β1, vimentin and collagen α1, whereas the addition of the TSG-6 antibody neutralized the inhibitory effect of TSG-6 on the activation of LX2 cells. In addition, cytoplasmic lipid drops, the marker of inactivated hepatic stellate cell, were detected in TSG-6-CM-cultured LX2 cells, only. The suppressed TSG-6 activity by TSG-6 antibody attenuated the restoration process in livers of TSG-6-CM-treated mice with CCl4. These results demonstrated that TSG-6 contributed to the liver regeneration by suppressing the activation of hepatic stellate cells in CCl4-treated mice, suggesting the therapeutic potential of TSG-6 for acute liver failure.